Process for the preparation of creatine phosphate

ABSTRACT

Process for preparing creatine phosphate comprising incubating in an alkaline medium creatine and 3-phosphoglyceric acid with an aqueous muscle extract in the presence of a catalytic amount of adenosine triphosphate, heating the resulting mixture for a short period of time to a temperature of 70* to 100* C. and recovering the creatine phosphate which is thereby formed.

United States ate-m Beaucamp 1 Febl ll M72 [54] PROQESS FOR THEPREPARATHON UN [56] References Cited REA'MNE PM SPHATE C U OTHERPUBLICATIONS [72] Inventor: Klaus Beaucamp Tumnglobb Germany Morrison etaL, Biochem. J. vol. 79 pages 433- 446 (196i) [73] Assignee: BoehringerMannheim Gmlblll, Mannheim- Waldhof, Germany Primary Examiner-Alvin E.Tanenholtz Filed: Nov 19M Attorney-Burgess, Dmklage & Sprung [21] Appl.No.1 792,182 [57] ABSTRACT Process for preparing creatine phosphatecomprising incubat- 30 Foreign A li gi Priority Data ing in an alkalinemedium creatine and 3-phosphoglyceric acid with an aqueous muscleextract in the presence of a cata- NOV. 29, 1967 Germany ..P 16 43 701.7lytic amount of adenosine triphosphate, heating the resumng mixture fora short period of time to a temperature of to [52] US. Cl ..l/29, 195/30and mcovering the creatine phosphate which is 51 int. (:1. ..Cl2d 113/06thereby f d [58] Field of Search ..l95/28, 29, 30

11 Claims, No Drawings PROCESS FOR THE PREPARATION OF CREATINE PHOSPHATEThis invention relates to a process for preparing creatine phosphate.More particularly this invention relates to a process for preparingcreatine phosphate in an economically feasible manner and in largeamounts.

It is in the prior art to prepare creatine phosphate by thephosphorylation of creatine with phosphoroxychloride (K. Zeile and G.Pawaz, Hoppe-Seylers Zeitschrift fur physiologische Chemie 256, 1931938)). The principal disadvantages of this known process are:

a. Poor yield.

b. The reaction conditions must be maintained rather precisely in orderto achieve even this low yield.

c. The method is not suitable for being carried out on a large scale, asthe Na3 P04 slurry that develops can neither be stirred nor cooled.

It is an object of this invention to provide a method of preparingcreatine phosphate which can be carried out in a simple and economicallyfeasible manner.

It is another object of the invention to provide a method of preparingcreatine phosphate in high yields and which is suitable for operation ona large scale.

These and other objects will become apparent from consideration of thedisclosure which follows.

In accordance with the invention, a method is provided for thepreparation of creatine phosphate in practically unlimited quantities inan extremely simple manner and in excellent yields which is based on theenzymatic phosphorylation of creatine. The method comprises incubatingcreating and 3- phosphoglyceric acid with an aqueous muscle extract inthe presence of a catalytic amount of adenosine triphosphate in analkaline medium, thereafter heating briefly the solution at atemperature of 70 to 100 C. and isolating the creatine phosphatetherefrom.

The enzymatic phosphorylation of creatine for the analytic determinationof creatine in tissues has already been described (H. U. Bergmeyer,METHODEN DER ENZY- MATISCHEN ANALYSE, Verlag Chemie, Academic Press,1963, p. 407). This process, however, could not be used for preparationof creatin phosphate as it requires the use of highly expensivephosphoenolpyruvate and highly purified creatine phosphokinase, andfurther the reaction is hampered by serum components. Furthermore, atthe concentrations which are required to be used, the isolation ofcreatine phosphate would be very expensive.

It was therefore very surprising to find that an enzymaticphosphorylation of creatine could be successfully carried out accordingto the method of the invention in an extremely simple manner withexcellent yields, and without any of the difficulties or disadvantagesdescribed above.

It is also surprising that the enzymatic reaction can be interrupted byheating to the stated temperature as from the teaching of Zeile et al.,it was to have been concluded that creatine phosphate is extremelysensitive to temperature, the chemical reaction being carried out at lowtemperature, or in any case at no higher than room temperature. Theacid-sensitivity of phosphocreatine also bars the conventional methodsof isolating organic phosphoric acid compounds, such as for example, themethods used in isolating nucleotides or sugar phosphates. Anyacidification, be it ever so brief, and any exchange chromatography orpassage through charcoal, results in considerable losses. Actually, ashas now been found, creatine phosphate can withstand as much as minutesof heating at 80 to 90 C, in the weakly alkaline range withoutperceptible losses.

The muscle extract which is used in the process of the invention can beeasily prepared by extraction of fresh meat with water, for example,rabbit or veal, and filtering offthe insoluble components. However,extracts of muscles of other origin can be used in place of the veal orrabbit extracts, with equal success. The important thing is for themuscle extract to contain a certain amount of creatine phosphokinase,although it may be very much less than the amount required according tothe analytical procedure. One unit of creatine phosphokinase for every 1to 10 mg. of creatine suffices for a satisfactory carrying out of theprocess of the invention. Larger amounts of the enzyme can, of course,be used; this results in a slight shortening of the time required forthe reaction, but other than this it offers no other special advantages.

The use of a buffer is not necessary. It is advantageous to addmagnesium and potassium ions if the concentration of these ions in thecrude muscle extract is substantially less than 1 to 2X10M. Increasingthe concentrations above these values results in no special advantages.

Creatine and 3-phosphoglyceric acid are utilized in approximatelyequimolar ratio in the reaction. The reaction can easily be carried outusing a slight excess of one of these two materials, until the completereaction of the other reagent has been realized. Preferably, about 1.1to 1.5 equivalents of creatine were used per equivalent of3-phosphoglyceric acid in order to achieve the complete reaction of thelatter. An excess of creatine is advantageous, as this is the:substantially less costly of the two reagents.

Adenosine triphosphate is added only in catalytic quantities. About 1equivalent of adenosine triphosphate is used for every 60 to 100equivalents of creatine or 3-phosphoglyceric acid, as the case may be.The amount of adenosine triphosphate can, of course, be increased,although this is not desirable for economic reasons and technically itis not particularly advantageous. The amount of adenosine triphosphatecan also be reduced, but this results in a longer reaction time.

As has already been indicated, the addition of buffer is not necessary.The desired alkaline pH can be obtained by adjustment with any alkalinesubstance, such as, for example, caustic soda solution.

The pH is advantageously maintained at between about 7.0 to l l, andpreferably between 8 and 9. The reaction temperature is desirably keptat between room temperature and about 45 C., preferably between 32 C.and 40 C. The temperature can be increased above the just indicatedrange when a relatively C.

The reaction time depends on the temperature and on the amount of crudeenzyme used. Using the preferred quantities of enzyme and usingtemperatures as stated above, the yield amounts to about to percent oftheory following a reaction time of 1 hour. It is possible to extend thereaction time to 3 and, if desired, even to 4 hours under the conditionsstated, to achieve a percent yield.

When the reaction has proceeded and the desired conversion obtained, itis stopped by rapidly heating the reaction mixture to a temperature of70 to 100 C, and preferably 80 to 90 C. Protein is thereby coagulatedand settles out, and is removed following cooling, by the conventionalmethods as for example by filtration.

The recovery of the creatine phosphate is carried out by separating theforeign ions, concentrating the solution, and thereafter precipitatingout the creatine phosphate by the addition of alcohol or some othermaterial that reduces its solubility in the solution, preferablyaccompanied by inoculation with creatine phosphate crystals.

The solution is then treated in a weakly alkaline pH range, preferablyat a pH of 8 to 8.5 with a cation exchanger balanced with sodium ions.Preferably it is passed through an exchanger column, after which it isconcentrated, and alcohol added until a clearly preceptible turbidityoccurs. Preferably the mixture is inoculated with creatine phosphate andallowed to stand for a period of time in the cold state. The crudecreatine phosphate crystalline mass that forms is then separated fromthe liquid phase and purified by one or two recrystallizations.

The following examples serve to explain the invention but are in nowiseto be taken as a limitation thereof.

EXAMPLE I a. Preparation of a rabbit muscle extract Rabbit muscle waschopped, homogenized and extracted with water. The pH of the resultantmixture was adjusted to 6.5 and heated for 15 minutes at 53 C.Precipitated protein was removed bycentrifugation. Ammonium sulfate wasadded to the supernatant liquid to provide a 1.5 M concentration. Anyprotein which was precipitated out was centrifuged off and ammoniumsulfate was added to the supernatant to provide a 3 M concentration andthe ammonium sulfate paste thereby obtained dialyzed for 3 hours againsttap water and used for the next reaction. b. Preparation of creatinephosphate Two hundred sixty g. of creatine were dissolved in 100 litersof water, and 15 g. of ATPNa,l-l -H O (corresponding to about 12 g. ofATP), 380 g. of 3-phosphoglyceric acid sodium, 14 g. of magnesiumacetate, 17 g. of potassium acetate and 80 g. of ammonium acetate werethan added to the solution and the resulting batch mixed thoroughly. ThepH was adjusted with soda lye to a value of 8.5 and the mixture heatedto 37 C. Thereafter the enzyme mixture prepared as set out in (a) abovewas added. This enzyme mixture contained about 80,000 units of creatinephosphokinase, about 100,000 units of enolase, about 100,000 units ofphosphoglycerate mutase and about 250,000 units of pyruvate kinase. Themixture was allowed to stand for 1 to 3 hours at 37 C. while maintainingthe pH value constant.

Following the completion of the reaction, which could be followed by theenzymatic analysis of the creatine phosphate which formed, the mixturewas heated rapidly to 80 to 90 C. and then cooled again. The proteinwhich precipitated out was filtered off, if desired, and the remainingsolution flowed through litres of cation exchanger (e.g. Amberlite IR120 or Dowex 50) which had been treated with sodium hydroxide and washedto provide a pH of 8-9. The yellowish clear liquid which emerged wascollected and concentrated until the volume amounted to about 4 to 6liters. Eight to 12 liters of alcohol were then added under agitation.When turbidity was observed to have set in, the mixture was inoculatedwith crystallized sodium creatine phosphate. The resulting mixture wasallowed to stand overnight in the cold, the precipitated crystallinemass suction filtered and thoroughly washed with alcohol. Afterrecrystallization the creatine phosphate was vacuum dried. The yieldamounted to 300 to 350 g. of creatine phosphate (COP-Na2-6H O).

EXAMPLE 2 The same procedure was followed as has been described inexample 1, using in place of the dialyzed crude rabbit enzyme, a vealextract, which was obtained by extracting freshly ground veal with twicethe volume of water and filtering off the solids. The quantity ofextract which was used was sufficient so as to supply 50,000 to 80,000units of creatine phosphate.

The addition of magnesium and potassium ions could be omitted in thiscase since a sufficient amount of such ions were present in the extract.

The reaction took from 2 to 4 hours, and the yield amounted to 250 to300 g. as further recrystallizations were necessary.

lclaim:

1. Process for preparing creatine phosphate as crystallized sodium saltwhich comprises incubating in an alkaline medium an aqueous muscleextract containing creatine phosphokinase in an amount sufficient tosupply one unit of creative phosphokinase for each 1 to 10 mg. creatinepresent with creatine and and 3-ph0sphoglyceric acid in the presence ofa catalytic amount of adenosine triphosphate, heating the resultingsolution for a short period of time to a temperature of 70 to C. andthereafter recovering the creatine phosphate as crystallized sodium saltthereby formed from said solution.

2. Process according to claim 1 which comprises utilizing 1.1 to 1.5equivalents of creatine per equivalent of 3- phos hoglyceric acid.

3. Process according to claim 1 which comprises utilizing said adenosinetriphosphate disodium salt and creatine in a molar ratio of 1:60 to1:100.

4. Process according to claim which comprises conducting said incubationat a pH of from 7.0 to l l.

5. Process according to claim 1 which comprises conducting saidincubation at a pH of 8.0 to 9.0.

6. Process according to claim 1 which comprises conducting saidincubation at a temperature ranging from room temperature to about 45 C.

7. Process according to claim 1 which comprises conducting saidincubation at a room temperature of from 32 to 40 C.

8. Process according to claim 1 which comprises utilizing as said muscleextract an aqueous veal extract.

9. Process according to claim 1 which comprises utilizing as said muscleextract an aqueous rabbit extract.

10. Process according to claim 9 which comprises preparing said aqueousrabbit muscle extract by extracting homogenized rabbit muscle withwater, heating the aqueous extract to a temperature of from about 50 to60 C, separating off any precipitated protein, introducing ammoniumsulfate into the supernatant liquid to provide a 3 M concentration,separating off any precipitated protein and dialyzing the same.

11. Process according to claim 1 which comprises adjusting theconcentration of magnesium and potassium ions in said aqueous muscleextract to a value of l-2X l0 for each of said ions.

. UNITED STATES PATENT OFFICE CERTIFICATE OF CURRECTION Patent No.3,639,211 Dated February 1, 1972 Inventor(s) Klaus Beaucamp It iscertified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below:

Col. 2, line 39 after "relatively" insert --hi.gh enzyme concentrationis used, and can even exceed by a slight amount 50C."

Col. 3, line 42 "(coP-uezmu fl) should be (CP-Na -6H 0) Claim 1, line 14Delete "and" (second occurrence) Claim 4-, line 1 After "claim" insert lSigned and sealed this 27th. day of February 1973..

'sEAL1 Attest;

EDWARD M.,FLETCHER,JR. ROBERT GOTTSGHALK Affpqfi'ng ()FfirprCOITllTllSSlOTlGI of Patents

2. Process according to claim 1 which comprises utilizing 1.1 to 1.5equivalents of creatine per equivalent of 3-phosphoglyceric acid. 3.Process according to claim 1 which comprises utilizing said adenosinetriphosphate disodium salt and creatine in a molar ratio of 1:60 to1:100.
 4. Process according to claim which comprises conducting saidincubation at a pH of from 7.0 to
 11. 5. Process according to claim 1which comprises conducting said incubation at a pH of 8.0 to 9.0. 6.Process according to claim 1 which comprises conducting said incubationat a temperature ranging from room temperature to about 45* C. 7.Process according to claim 1 which comprises conducting said incubationat a room temperature of from 32* to 40* C.
 8. Process according toclaim 1 which comprises utilizing as said muscle extract an aqueous vealextract.
 9. Process according to claim 1 which comprises utilizing assaid muscle extract an aqueous rabbit extract.
 10. Process according toclaim 9 which comprises preparing said aqueous rabbit muscle extract byextracting homogenized rabbit muscle with water, heating the aqueousextract to a temperature of from about 50* to 60* C, separating off anyprecipitated protein, introducing ammonium sulfate into the supernatantliquid to provide a 3 M concentration, separating off any precipitatedprotein and dialyzing the same.
 11. Process according to claim 1 whichcomprises adjusting the concentration of magnesium and potassium ions insaid aqueous muscle extract to a value of 1-2 X 10 3for each of saidions.